The long-term goal of Dr. Bradbury's laboratory is to understand the mechanism of endocytosis of ion channels, using endocytosis of the cystic fibrosis transmembrane conductance regulator (CFTR) as an experimental system. CFTR is important in the regulation of chloride secretion by the cAMP-mediated second messenger cascade, and mutations in CFTR give rise to the human genetic disease cystic fibrosis (CF). Our working model predicts that cAMP not only increases the open probability (Po) of CFTR, but also regulates the number (N) of CFTR molecules in the plasma membrane. Endocytosis of CFTR is a major mechanism for acutely regulating the distribution of CFTR between the plasma membrane and subcellular organelles such as endosomes and recycling vesicles. Therefore endocytosis may partly determine the magnitude of the secretory response to cAMP mediated agonists. However, the mechanisms of the specific steps of CFTR endocytosis are not yet fully understood. Proposed research will focus on the steps and molecular interactions necessary for the endocytosis of CFTR. The emphasis of the project will be on the search for and characterization of clathrin adaptor molecules that allow effective interaction of CFTR with a plasma membrane clathrin coated pit, a key element of the endocytic apparatus. The general strategy of these studies falls into two related directions. We will determine which sites on CFTR directly bind to plasma membrane adaptor proteins, and determine which subunits of the adaptor proteins are responsible for this interaction. Identification of binding sites for adaptor proteins should provide a basis for understanding the mechanisms of specificity in CFTR endocytosis. We will analyze the role of CFTR phosphorylation in this interaction, to determine if phosphorylation status of CFTR modulates its affinity for adaptor proteins and hence efficiency of endocytosis. These studies will be accompanied by a kinetic analysis of CFTR endocytosis in cells expressing mutants of CFTR which lack plasma membrane adaptor binding motifs. Similarly the endocytic kinetics of CFTR phosphorylation mutants will be evaluated in the presence and absence of cAMP raising agents. Studies of the dynamics of CFTR internalization may, in turn, help identify other proteins regulating CFTR endocytosis. Results from our studies should allow us to design experimental approaches to prevent the endocytosis of CFTR mutants that reach the plasma membrane but have a low open probability upon activation.